Download passive micromechanical properties of isolated human muscle fiber

PASSIVE MICROMECHANICAL PROPERTIES OF ISOLATED HUMAN MUSCLE FIBER SEGMENTS
+*Lieber, R (A-VA and NIH); *Jordan, K; **Fridén, J (A-Swedish Medical Research Council)
+*V.A.M.C. and University of California, San Diego, La Jolla, CA. 9500 Gilman Drive, La Jolla, CA 92039-9151, 858-552-8585, Fax: 858-552-4381,
[email protected]
INTRODUCTION: Surgical tendon transfer is commonly used to restore
lost motor function after stroke, head injury or trauma [1]. Intraoperative
manipulation of human skeletal muscle is based primarily upon surgeon’s
“feel” for the tissue during the procedure. It has been demonstrated that
during such procedures, very long, even nonfunctional sarcomere lengths are
often imposed [2]. It is difficult to interpret the relationship between passive
force and sarcomere length in whole muscles due to the presence of
intracellular structural proteins, extracellular matrix, connective tissue
components and gross fascial connections to adjacent muscles. In order to
establish the passive properties of human muscle fibers absent other
complicating mechanical components, the purpose of this study was to
measure the relationship between passive stress and sarcomere length in
isolated human single fiber segments.
METHODS: After informed consent and incidental to other surgical
procedures such as fracture repair or plate removal, small (3 mm x 10 mm)
muscle biopsies were obtained with special care to orient the biopsy along the
muscle fiber axis (n=10 fibers from 6 biopsies). Biopsies were immediately
transferred to a high potassium, low sodium ion concentration “relaxing”
solution to prevent mechanical disruption and hypercontraction [3]. Single
fiber segments were then microdissected from the biopsy and transferred to a
custom chamber where they were secured to a microforce transducer (Aurora
Scientific Model 405, sensitivity 10V/g) and high resolution motor (Aurora
Scientific Model 300B, sensitivity 3V/mm) using 10-0 suture loops (Fig. 1).
RESULTS: Using the dissecting procedure developed in the relaxing
solution, it was fairly easy to obtain intact fiber segments that ranged from 2-8
mm in length. In some cases, it was not possible to obtain intact fiber
segments due to iatrogenic trauma during biopsy removal, inadvertent trauma
during dissection, or disruption during mounting. In any case, fibers that were
disrupted lost their translucent appearance, developed size abnormalities along
their length and did not yield clear diffraction patterns. We are confident that
all fibers reported here were intact. Average “slack” sarcomere length of the
fibers was 2.25 ±0.15µm (n=10) which was significantly less than the
estimated muscle optimal sarcomere length of 2.6 µm determined by onesampled test (p<0.001, Fig. 2). The distribution of lengths was nonnormal,
clustering at 2.0 and 2.5 µm. Not surprisingly, fiber stress increased with
increasing fiber length (Fig. 3) and based on the average fiber size of
2853±357 µm2, this corresponded to a peak fiber stress of only 25.5±4.4 kPa.
FIGURE 3: Sample relationship between sarcomere length and fiber stress in
an isolated human fiber segment. Solid line represents the hypothetical
descending limb of the human length-tension curve which begins at sarcomere
length of 2.7 µm and 250 kPa and ends at sarcomere length 4.2 µm and 0 kPa.
FIGURE 1: Single fiber segment attached to force transducer and motor arm
via 125 µm titanium wire viewed through dissecting microscope. Fiber
diameter is approximately 40 µm. Calibration bar is 0.5 mm.
The single fiber segment was transillumated by a 7 mW He-Ne laser to permit
sarcomere length measurement by laser diffraction [4]. The fiber segment
was set to “slack length” which was defined as the length where (1) the
diffraction pattern was just oriented along the fiber axis, (2) tension remained
within ±1 standard deviation (SD) of the noise level of the transducer (10 mV)
and (3) the fiber did not have a wavy appearance. In practice, this was easily
reproducible. Fibers were lengthened in 250 µm increments for 2 mm and
500 µm increments thereafter. Sarcomere length and tension were measured 1
minute after each length change to permit fiber stress-relaxation.
FIGURE 2: Mean slack sarcomere length
measured from 10 isolated single fiber
segments. Individual data points are
shown by circles and mean±SD by bar.
Note clustering of slack sarcomere lengths
around 2.0 and 2.5 µm. Slack sarcomere
length represents the length that the
sarcomere naturally assumes under zero
load. This is related but not identical to
the muscle “resting length.”
DISCUSSION: This study reports the first values for slack sarcomere length
and sarcomere length-stress relations for human skeletal muscle cells. These
data are extremely valuable not only for understanding the biomechanical
basis for surgical procedures, but for modeling musculoskeletal function.
Heretofore, human passive mechanical data were simply extrapolated from
other mammalian literature. The short slack sarcomere length of 2.25 µm was
unexpected considering that human myofilaments are considerably longer
compared to other animals [5]. This result suggests that, if passive
mechanical properties of human muscles measured intraoperatively reflect
muscle fiber properties, human fibers would be very vulnerable to overstretch.
Interestingly, this phenomenon has been recently reported [2]. The structural
basis for the very low stiffness and bimodal distribution of slack sarcomere
lengths in these fibers is likely to be the intracellular cytoskeletal protein
known as titin [6].
REFERENCES
**Sahlgrenska University Hospital, Göteborg, SWEDEN.
0716
Poster Session - Muscle and Nerve - Hall E
47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California